WO2023115347A1

WO2023115347A1 - Fully human antibody targeting gprc5d

Info

Publication number: WO2023115347A1
Application number: PCT/CN2021/140153
Authority: WO
Inventors: 谭涛超; 魏巧娥; 贾向印; 黄星星; 张千千; 刘建伟; 谢萌; 金亮
Original assignee: 上海驯鹿生物技术有限公司
Priority date: 2021-12-21
Filing date: 2021-12-21
Publication date: 2023-06-29

Abstract

Provided is a fully human antibody targeting GPRC5D, or an antigen-binding fragment thereof, comprising a fully human antibody, which specifically binds to GPRC5D with a high affinity. Further provided are a fusion protein, bispecific antibody and immunoconjugate comprising the fully human antibody targeting GPRC5D, or the antigen-binding fragment thereof, and the use thereof in detection and treatment.

Description

Fully human antibody targeting GPRC5D

technical field

The present invention relates to an antibody targeting GPRC5D, especially a fully human antibody targeting GPRC5D. The present invention also relates to the application of the antibody.

Background technique

Multiple myeloma (multiple myeloma) is a plasma cell tumor that occurs in the bone marrow. The tumor causes conditions such as hypercalcemia, anemia, renal dysfunction, osteonecrosis, and bone marrow failure. Multiple myeloma is currently the second most common hematological malignancy. According to the statistics on the incidence of multiple myeloma released by the World Health Organization (WHO) in 2020, the morbidity and mortality of the Asian population accounted for 36% and 42%, respectively, ranking first in the statistics of all continents. The morbidity and mortality ratio of multiple myeloma in the world is 1.8:1.1, and it is 1.1:0.76 in Asia. It can be seen that the survival rate of multiple myeloma in the affected population is low. The main reason is that the median age of the population is about 66 years old, and the incidence rate of people under the age of 40 is about 2%. The second is that multiple myeloma has almost no cure possibility under the commonly used chemotherapy, immunomodulators and monoclonal antibody therapy, and barely remission is accompanied by a very high recurrence rate, that is, relapsed/refractory multiple myeloma (Relapsed/refractory Multiple myeloma (Relapsed/refractory Multiple myeloma) RRMM), the five-year survival rate is only 51%.

In recent years, T cell bispecific antibody therapy (bispecific T cell engagers, BiTEs) and secondary T cell therapy (adoptive T cell therapy, ACT) have made breakthroughs in the treatment of multiple myeloma. Targeted therapy products for multiple myeloma mainly target BCMA, CD38, CD138, GPRC5D, etc. CD38 and CD138 are also expressed on cells of normal tissues and hematopoietic stem cells. After targeted therapy is used to eliminate them, side effects are relatively large, and they often cause damage to normal organs or damage the autoimmune system. BCMA and GPRC5D are mainly expressed in plasma cells or plasma cells in myeloma, which can be replenished by the continuous regeneration of the body's own B cells. Taking T cell bispecific antibody products as an example, products targeting BCMA mainly include REGN5458 developed by Regeneron Pharmaceuticals, Inc. (REGN), Teclistama developed by Johnson & Johnson, and AMG420 developed by Amgen. REGN5458 has completed Phase I clinical trials (NCT03761108). Of the 19 patients receiving treatment, 42% achieved complete remission (CR) or strict complete remission (sCR); Teclistamab has entered the clinical phase II trial (NCT04557098), and the clinical phase I results show that the overall response rate (ORR) is 65% , 40% of patients achieved complete remission (CR); AMG420 clinical phase I (NCT03836053) showed an overall response rate (ORR) of 71%.

The CAR-T product developed by Bristol-Myers Squibb (BMS) targeting the BCMA target of multiple myeloma - bb2121 was approved by the FDA in May 2021 as the first CAR-T product for multiple myeloma to be marketed. The product's overall response rate (ORR) was 72%, and 28% of patients achieved strict complete remission (sCR). Although this product has brought hope for the cure of multiple myeloid, according to the published data, the 22-month progression-free survival rate of patients with complete remission (CR) is less than 50%, which shows that the late stage of treatment There is also a high recurrence rate. In addition, since the therapy eliminates all plasma cells, there are still side effects caused by decreased blood cells and immunoglobulins. The expression of GPRC5D is more specific than that of BCMA, and it is only expressed in plasma cells of myeloma patients, while it is almost not expressed in normal tissues, and significant RNA and protein expression can only be detected in hair follicle tissues. No significant difference was found in the phenotype (including body weight, organ morphological differences, reproductive rate, etc.) between GPRC5D-knockout mice and normal wild-type mice. It can be seen that GPRC5D deletion is not necessary for survival and normal organ metabolism, and its clearance less side effects. It was further found that the expression of BCMA was not correlated with GPRC5D. Although both were expressed in plasma cells at the same time, their expression profiles were relatively independent. It can be seen that the expression of BCMA in patients with BCMA CAR-T therapy in the late stage is low, and GPRC5D can be targeted for treatment.

Therefore, the development of fully human antibodies that can exert clinically effective cytotoxic, cytostatic or immunosuppressive effects on cells expressing GPRC5D, and have no adverse effects on cells that do not express GPRC5D, is very useful for the development of immunotherapy products related to GPRC5D expression. Significance.

Contents of the invention

In one aspect, provided herein is a GPRC5D binding peptide comprising a heavy chain variable region of an antibody molecule, wherein HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are selected from one of the following combinations:

1) HCDR1 is GGSFSGYY (SEQ ID NO: 1);

HCDR2 is INHSGST (SEQ ID NO: 2);

The sequence of HCDR3 is ARARRYGGRTRFDP (SEQ ID NO: 3);

2) HCDR1 is GFIFSSYG (SEQ ID NO: 4);

The sequence of HCDR2 is ISSSGDYT (SEQ ID NO: 5);

The sequence of HCDR3 is ARMSFRRYDH (SEQ ID NO: 6); and

3) HCDR1 is GFSFSGYI (SEQ ID NO: 7);

The sequence of HCDR2 is TSSSGTET (SEQ ID NO: 8);

The sequence of HCDR3 is ARYYSKYGRSYHVDS (SEQ ID NO: 9).

In some embodiments, the GPRC5D-binding peptide is an antibody molecule or an antigen-binding fragment thereof.

In some embodiments, the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 10, 11 or 12; or the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 10, 11 or 12 The amino acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity and is capable of specifically binding GPRC5D.

In some embodiments, the GPRC5D binding peptide is a single domain antibody molecule.

In some embodiments, the GPRC5D-binding peptide is a fully human antibody molecule.

In some embodiments, the binding ability of the GPRC5D-binding peptide to cells expressing GPRC5D on the surface is that the EC50 value detected by FACS is no higher than 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, or 5 nM.

In another aspect, fusion proteins are provided herein that include the GPRC5D-binding peptides described above.

In some embodiments, the fusion protein includes at least two GPRC5D binding peptides.

In some embodiments, said HCDR1, HCDR2 and HCDR3 of said two GPRC5D binding peptides are respectively selected from the different combinations listed above.

In some embodiments, in the fusion protein: one of the two GPRC5D binding peptides includes the amino acid sequence shown in SEQ ID NO: 10, or includes at least 90%, 91% of the sequence shown in SEQ ID NO: 10 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity amino acid sequence and can specifically bind to GPRC5D, another comprising SEQ ID NO: 11 shown Amino acid sequence, or includes at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence shown in SEQ ID NO: 11 amino acid sequence and can specifically bind GPRC5D; one of the two GPRC5D binding peptides includes the amino acid sequence shown in SEQ ID NO: 10, or includes at least 90%, 91%, 92% of the sequence shown in SEQ ID NO: 10 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity amino acid sequence and can specifically bind GPRC5D, another amino acid sequence comprising SEQ ID NO: 12, or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence shown in SEQ ID NO: 12 and Able to specifically bind GPRC5D; or one of the two GPRC5D binding peptides includes the amino acid sequence shown in SEQ ID NO: 11, or includes at least 90%, 91%, 92%, An amino acid sequence with 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity and capable of specifically binding to GPRC5D, another amino acid sequence comprising SEQ ID NO: 12, or comprising An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 12 and capable of specific Sexually binds GPRC5D.

In some embodiments, the fusion protein includes at least three of the GPRC5D-binding peptides, wherein the HCDR1, HCDR2 and HCDR3 of the three GPRC5D-binding peptides are the three combinations listed above, respectively.

In some embodiments, the first of the three GPRC5D-binding peptides of the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 10, or comprises at least 90% of the sequence set forth in SEQ ID NO: 10 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity amino acid sequence and can specifically bind GPRC5D; the second comprises SEQ ID NO: 11 The amino acid sequence shown, or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the sequence shown in SEQ ID NO: 11 Consistent amino acid sequence and can specifically bind GPRC5D; and the third comprises the amino acid sequence shown in SEQ ID NO: 12, or comprises at least 90%, 91%, 92%, An amino acid sequence having 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity and capable of specifically binding to GPRC5D.

In some embodiments, the fusion protein also includes an Fc fragment.

In some embodiments, the fusion protein further comprises another binding peptide capable of specifically binding an antigen other than GPRC5D.

In some embodiments, the other binding peptide is a single domain antibody or a single chain antibody.

In some embodiments, the fusion protein further includes a detectable tag or a purification tag.

In some embodiments, the detectable label is enzymatically active.

In another aspect, provided herein are nucleic acid molecules encoding the above-mentioned GPRC5D-binding peptide or the above-mentioned fusion protein.

In some embodiments, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 13, 14 or 15.

In another aspect, provided herein are expression vectors comprising the nucleic acid molecules described above.

In another aspect, provided herein are host cells comprising the nucleic acid molecules or expression vectors described above.

In another aspect, this paper provides a multispecific antibody molecule, which at least includes a first functional part and a second functional part, wherein the first functional part includes the above-mentioned GPRC5D binding peptide; the second functional part has a different binding specificity.

In some embodiments, the second functional moiety has binding specificity for immune cells.

In some embodiments, the second functional moiety has binding specificity for T cells.

In some embodiments, the second functional moiety has binding specificity for CD3.

In another aspect, provided herein are immunoconjugates comprising the above-described GPRC5D-binding peptides linked to a therapeutic agent.

In some embodiments, the therapeutic agent is a drug.

In some embodiments, the therapeutic agent is a cytotoxin.

In some embodiments, the therapeutic agent is a radioisotope.

On the other hand, this paper provides a pharmaceutical composition, which includes: 1) the above-mentioned GPRC5D-binding peptide; the above-mentioned fusion protein; the above-mentioned multispecific antibody molecule; or the above-mentioned immunoconjugate; and 2) a pharmaceutically acceptable carrier.

The above-mentioned GPRC5D-binding peptides, fusion proteins, multispecific antibody molecules, immunoconjugates or pharmaceutical compositions can be used to treat GPRC5D-related diseases.

In another aspect, this paper provides the use of the above-mentioned GPRC5D-binding peptide, fusion protein, multispecific antibody molecule or immunoconjugate in the preparation of a medicament for treating GPRC5D-related diseases.

In some embodiments, the GPRC5D-associated disorder is cancer, or an autoimmune disease, and the cancer is preferably a plasma cell malignancy, such as multiple myeloma, or a B cell malignancy, such as Hodgkin's lymphoma or non-Hodgkin's lymphoma. Chiggin's lymphoma.

In another aspect, provided herein is a method for treating GPRC5D-related disorders, which comprises administering an effective amount of the above-mentioned GPRC5D-binding peptide, fusion protein, multispecific antibody molecule, immunoconjugate or pharmaceutical composition to a subject in need medicine.

In another aspect, this paper provides a method for detecting the content of GPRC5D in a biological sample, comprising:

1) contacting said biological sample with said GPRC5D-binding peptide, fusion protein or multispecific antibody molecule to form a binding complex with said GPRC5D;

2) Determining the content of GPRC5D in the biological sample based on the amount of the binding complex generated in step 1).

1) coupling the above-mentioned GPRC5D-binding peptide to a detectable label;

2) contacting said GPRC5D-binding peptide bearing said detectable label with said biological sample;

3) determining the content of GPRC5D in the biological sample by detecting the amount of the detectable label.

In another aspect, a detection kit is provided herein, which includes the above-mentioned GPRC5D-binding peptide, fusion protein or multispecific antibody molecule.

In another aspect, provided herein is a pharmaceutical kit comprising the above-mentioned GPRC5D-binding peptide, fusion protein or multispecific antibody molecule.

In another aspect, provided herein are methods of identifying a GPRC5D-associated disorder in a subject, comprising:

1) using the above detection kit to determine the content of GPRC5D in the biological sample from the subject; and

3) Compared with the normal GPRC5D content in the population to determine whether the GPRC5D-related disease exists or its status.

In another aspect, this paper provides a method for determining the therapeutic effect of a drug for treating a GPRC5D-related disorder in a subject, comprising:

1) Using the above method to identify the GPRC5D-related disease state of the subject;

2) treating the subject with the drug;

3) Repeat step 1) to determine the change of the GPRC5D-related disease state in the subject, and determine the therapeutic effect of the drug based on the change.

Description of drawings

Figure 1 shows the enzyme-linked immunosorbent assay (ELISA) results of part of the panned phage monoclonals, target antigens and control antigens.

Figure 2A shows the results of flow cytometric analysis of CHO-K1-GPRC5D cell line stained with different antibodies; Human GPRC5D PE-conjugated Antibody stained is the result of commercialized flow cytometric antibody staining, Fluorescein (FITC) AffiniPure Goat Anti-Human IgG stained It is the negative control stained with fluorescently labeled antibody only; Positive benchmark stained/Fluorescein (FITC) AffiniPure Goat Anti-Human IgG stained is the result of staining with positive control antibody and fluorescently labeled antibody; Figure 2B shows some phage monoclonal and CHO - Flow cytometric analysis results of K1-GPRC5D and CHO-K1 cell binding.

Figure 3 shows the results of ELISA analysis of the screened phage monoclonals at the phage level and various non-related antigens. Negative phage control is a negative control phage antibody clone, Anti-M13 phage mouse/anti-mouse HRP Ab is a negative antibody control with only primary and secondary antibodies added, anti-mouse HRP Ab is a negative antibody control with only secondary antibodies added, anti -human IgG HRP Ab is a negative antibody control with secondary antibody added only, anti-his tag HRP Ab is a positive antibody control for detecting antigen tags, and Positive Benchamrk1 is a positive antibody control for target antigen.

Figure 4 shows the results of flow cytometric analysis of the binding of the screened phage monoclonals to various GPRC5D positive and negative cell lines at the phage level. Negative phage Control is a negative control phage antibody clone, Positive Benchamrk1 Ab is a positive antibody to the target antigen, and FITC anti-human IgG Ab is a negative control with only secondary antibodies added.

Figure 5 shows the results of ELISA analysis of the screened phage monoclonals at the protein level and various non-related antigens. anti-human IgG HRP Ab is a negative antibody control with secondary antibody added only, anti-his tag HRP Ab is a positive antibody control for detecting antigen tags, and Positive Benchamrk1 is a positive antibody control for target antigen.

Figure 6 shows the results of flow cytometric analysis of the binding of the screened phage monoclonals to various GPRC5D positive and negative cell lines at the protein level. Positive Benchamrk1 Ab is a positive antibody to the target antigen, and FITC anti-human IgG Ab is a negative control with only secondary antibodies added.

Figure 7 shows the results of FACS detection of the binding of the monoclonal to the cell CHO-K1-GPRC5D highly expressing the target antigen.

Detailed ways

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

Antibodies refer to immunoglobulins secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (polypeptides, viruses, bacteria, etc.). This foreign substance is accordingly called an antigen. The basic structure of a classical antibody molecule is a 4-mer composed of 2 identical heavy chains and 2 identical light chains. According to the conservative difference in amino acid sequence, the heavy chain and light chain are divided into variable region (V) located at the amino terminal and constant region (C) located at the carboxy terminal. The variable regions of one heavy and one light chain interact to form the antigen binding site (Fv). In the variable region, the composition and arrangement of amino acid residues in certain regions are more variable than other regions (framework regions, FRs) in the variable region, which are called hypervariable regions (HVR), and the hypervariable regions are actually antibodies. Key site for antigen binding. Since these hypervariable regions are complementary to antigenic determinants, they are also called complementarity-determining regions (CDRs). Both the heavy and light chains have three complementarity determining regions, called HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, respectively.

The "antigen-binding fragment" of an antibody molecule refers to an amino acid fragment in an antibody molecule that participates in antigen-specific binding, for example, Fab, Fab' and (Fab') ₂ and the like. Single-chain antibodies and single-domain antibodies with antigen-binding ability are single-peptide chain antibody molecules, which can be regarded as "antigen-binding fragments" of classical antibody molecules.

"Fc fragment" refers to the handle region of the Y"-shaped antibody molecule, that is, the fragment crystallizable (Fc) includes the second and third constant domains (CH2 and CH3 domains) of the heavy chain. (eg, papain) hydrolysis of the antibody molecule yields the Fc region of the antibody. In some examples, the Fc region may comprise a hinge, CH2, and CH3. When the Fc region comprises a hinge, it may mediate dimerization between two Fc-containing polypeptides. The Fc fragment can be from IgG, IgM, IgD, IgE, or IgA. In some examples, the Fc region is from IgGl, IgG2, IgG3, or IgG4. "Fc fragment" also includes those derived from native Fc fragments that have been altered but retain their effector functions. Variant Fc fragments. A "variant Fc fragment" comprises an amino acid sequence having at least one amino acid change over the amino acid sequence of a native Fc fragment. In some instances, a variant Fc fragment has At least one amino acid substitution, for example about 1 to about 10 amino acids is substituted in the parental Fc fragment, and preferably about 1 to about 5 amino acid substitutions. In some instances, the variant Fc fragment Fc region has at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. The effector function of the "Fc fragment" can be Including binding to Fc receptors, Clq binding and complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), mediating phagocytosis, etc.

Single-chain antibody (single chain fragment variable, scFv) is composed of antibody heavy chain variable region and light chain variable region connected by a short peptide to form a peptide chain. Through correct folding, the variable regions from the heavy chain and the light chain interact through non-covalent bonds to form Fv segments, so scFv can better retain its affinity activity for antigens.

"Single domain antibody (single domain antibody, sdAb)", or also known as "VHH antibody", refers to an antibody molecule with antigen-binding ability, including a heavy chain variable region without a light chain. From a structural point of view, single-domain antibodies can also be considered as an antigen-binding fragment of an antibody molecule. It was first discovered in camelids. Subsequently, researchers screened antibody libraries (such as phage display libraries) and discovered more single-domain antibodies with antigen-binding ability. Single-domain antibodies have some advantages over ordinary antibody molecules (for example, classical tetrameric antibody molecules) or their antigen-binding fragments, including but not limited to: smaller molecular weight, making it easy to reach tissues that are difficult for ordinary antibody molecules to reach when used in the human body or parts, or can access antigenic epitopes in proteins or polypeptides that are difficult for ordinary antibody molecules to access; more stable, able to withstand changes in temperature and pH, as well as the action of denaturants and proteases.

A "fully human antibody" refers to an antibody in which both the variable and constant regions, if any, are derived from human germline immunoglobulin sequences. Fully human antibodies can be obtained by a variety of techniques, including phage antibody library technology, single B cell cloning technology, transgenic mouse technology (for example, using human germline immunoglobulin genes and removing the mouse's own germline immunoglobulin protein gene transgenic mice), etc. Compared with animal-derived antibodies (such as mouse-derived antibodies), fully human-derived antibodies have the advantages of less immunogenicity and higher safety when used in human patients.

"Bispecific antibody" refers to an antibody molecule that has two different binding sites and can recognize and bind two different antigens, respectively. For example, one binding site of a bispecific antibody can be used to bind immune cells (such as T cells), and the other binding site can be used to bind tumor cells, thereby enhancing the killing effect of immune cells on tumor cells while reducing side effects such as off-target toxicity . Such bifunctional antibodies usually have a higher efficacy than monoclonal antibody drugs as a drug for treating tumors. Similarly, antibody molecules can be engineered to include multiple distinct binding sites, resulting in "trispecific antibodies," "tetraspecific antibodies," and the like. As used herein, "multispecific antibody" encompasses such bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and the like.

"Immunoconjugate" refers to a GPRC5D binding peptide disclosed herein conjugated to a therapeutic agent. The treatment is, for example, cytotoxins, drugs (eg, immunosuppressants) or radiotoxins.

"Fusion protein" refers to a protein molecule that is artificially produced (for example, through genetic engineering techniques) and consists of at least two different peptide segments. These peptides do not exist in nature, or do not exist in the same protein molecule. Examples of common fusion proteins that include antibody fragments include antibody-cytokine fusion proteins, antibody-cytotoxin fusion proteins (also known as immunotoxins), enzyme-labeled antibodies for immunoassays, chimeric antigen receptors (CAR), etc. .

"Targeting" or "specific binding" refers to the fact that one molecule (such as an antibody or antigen-binding fragment thereof) has a higher affinity for another molecule (such as a tumor cell surface antigen) relative to other molecules that are also present in the environment. binding affinity. "Targeting" or "specifically binding" does not exclude that the molecule may have binding affinity for more than one molecule, eg a bispecific antibody may have high affinity for two different antigens.

"Binding complex" refers to a complex formed between a molecule and its binding partner that includes both. Examples of binding complexes may include antigen-antibody complexes, ligand-receptor complexes, protein dimers, and the like. The forces that form the binding complex mainly include non-covalent bond forces such as hydrogen bonds, van der Waals forces, and ionic bonds.

GPRC5D is a G protein-coupled receptor C5 family subtype D, which belongs to an orphan receptor and is a 7-transmembrane protein. Orphan receptors refer to receptors that are structurally similar to other recognized receptors, but their endogenous ligands have not been found yet. GPRC5D is highly expressed on the surface of primary multiple myeloma cells, while its expression in normal tissues is limited to the hair follicle area. Studies have shown that 65% of multiple myeloma patients have an expression threshold of GPRC5D exceeding 50%. With this feature, GPRC5D has become A potential target for the treatment of multiple myeloma (MM).

"Disease status" refers to whether the disease exists, the severity of the disease, the stage, type, and progress of the disease, etc.

GPRC5D-binding peptide refers to a polypeptide that targets or specifically binds to GPRC5D. For example, the GPRC5D binding peptide is a single domain antibody targeting GPRC5D.

"Peptide segment" refers to a polypeptide fragment, which is a short amino acid sequence, such as about 2-20 amino acids in length, and is usually a part of a polypeptide or protein.

EC50 (concentration for 50% of maximal effect) refers to the concentration that causes 50% of the maximal effect. When used in FACS to indicate the binding ability of antibody molecules to corresponding antigens on cells, it can refer to the concentration of antibody molecules that produces half of the maximum fluorescence intensity. The lower the EC50 value, the greater the binding affinity to the antigen on the cell.

"Purification tag" refers to the amino acid sequence used to purify the target protein or polypeptide expressed as a fusion protein with the target protein or polypeptide, including but not limited to His6 tag, Flag tag, MBP (maltose binding protein) tag and GST (gluten Glutathione thiol transferase) tag, SUMO (small ubiquitin related modifier (small ubiquitin related modifier)), etc. These tags can be removed by enzyme cleavage after purification, or can be used with tags (such as His6 tags) without affecting the normal function of the target protein or polypeptide.

"Detectable label" refers to an amino acid sequence or other chemical group attached to a protein or polypeptide to indicate the presence or amount of the protein or polypeptide in a sample, or to track the protein or polypeptide in the body or cells of a subject location information. Examples of detectable labels include various enzymes that can be used in immunoassays, such as horseradish peroxidase (HRP), alkaline phosphatase (ALP); fluorophores (such as FAM, FITC) or fluorescent proteins (such as GFP ); radioactive isotopes (eg ³ H, ¹⁴ C, ³⁵ S). When the detectable label is an enzyme, the presence or amount of the protein or polypeptide linked to the enzyme can be determined by the enzymatic activity of the enzyme.

The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues. Such polymers of amino acid residues may contain natural or unnatural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed within this definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for the purposes of the present invention, "polypeptide" refers to a protein that includes modifications to the native sequence, such as deletions, additions and substitutions (often conservative in practice), so long as the protein retains the desired activity. These modifications may be purposeful, such as induced through site-directed mutagenesis, or may be accidental, such as through mutation of the protein-producing host or errors due to PCR amplification.

Herein, the terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" are used interchangeably to refer to a polymer of nucleotides. Such nucleotide polymers may contain natural and/or unnatural nucleotides and include, but are not limited to, DNA, RNA and PNA. "Nucleic acid sequence" refers to the linear sequence of nucleotides comprised in a nucleic acid molecule or polynucleotide.

The term "vector" refers to a nucleic acid molecule (eg, nucleic acid, plasmid, or virus, etc.) that can be engineered to contain a polynucleotide of interest (eg, a coding sequence for a polypeptide of interest) or that can replicate in a host cell. A vector may include one or more of the following components: an origin of replication, one or more regulatory sequences (such as a promoter and/or enhancer) that regulate the expression of a polynucleotide of interest, and/or one or more Selectable marker genes (such as antibiotic resistance genes and genes useful in colorimetric assays, eg β-galactose). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

A "host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic or eukaryotic. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, 293, and CHO cells, and derivatives thereof, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complementation) to the original parent cell due to natural, accidental or deliberate mutation. Host cells also include cells transfected in vivo with nucleic acid molecules or expression vectors provided herein.

"Treatment" means manipulating a subject to obtain a beneficial or desired clinical result. "Treatment" as used herein encompasses a variety of manipulations including administration of any possible drug to a subject, surgery, radiation, and the like. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, attenuation of disease extent, prevention or delay of disease spread (e.g. metastasis, e.g. metastases to the lungs or lymph nodes), preventing or delaying disease recurrence, delaying or slowing down disease progression, improving disease conditions, inhibiting disease or disease progression, arresting its development and remission (whether partial or complete). The methods provided herein encompass any one or more of these therapeutic aspects. In the light of the above, "treating" does not require complete removal of all symptoms or complete remission of a disorder or disease.

The term "therapeutically effective amount" refers to the amount of an active compound sufficient to elicit a clinician's desired biological or medical response in a subject. The "therapeutically effective amount" of the fusion protein of the present invention can be determined by those skilled in the art according to factors such as administration route, subject's body weight, age, and condition. For example, a typical daily dosage may range from 0.01 mg to 100 mg or more of active ingredient per kg body weight.

In reference to pharmaceutical compositions, the term "pharmaceutically acceptable carrier" as used refers to substances such as solid or liquid diluents, fillers, antioxidants, stabilizers, etc. that can be safely administered, which are suitable for human and/or Animal administration without undue adverse side effects, while being suitable for maintaining the viability of the drug or active agent located therein. Depending on the route of administration, various carriers well known in the art can be administered, including, but not limited to, sugars, starches, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols , alginic acid, phosphate buffer, emulsifier, isotonic saline, and/or pyrogen-free water, etc. The pharmaceutical composition provided herein can be made into clinically acceptable dosage forms such as powder and injection. The pharmaceutical composition of the present invention may be administered to a subject by any suitable route, for example, orally, intravenously, intramuscularly, subcutaneously, subperitoneally, rectally, sublingually, or by inhalation, transdermally, etc. route of administration.

"Subject" means an animal, such as a mammal, including, but not limited to, a human, rodent, simian, feline, canine, equine, bovine, porcine, sheep, goat, lactating laboratory animals, mammalian farm animals, mammalian sports animals and mammalian pets. The subject can be male or female and can be of any appropriate age, including infant, infant, adolescent, adult and geriatric subjects. In some instances, a subject refers to an individual in need of treatment for a disease or condition. In some examples, a subject receiving treatment can be a patient who has, or is at risk of developing, a disorder associated with the treatment. In certain instances, the subject is a human, such as a human patient. The term is often used interchangeably with "patient", "subject", "subject" and the like.

As used herein, "population" generally refers to a healthy population. In the context of a particular analysis of a disease, "population" can also refer to individuals who do not have that disease but have other diseases. In addition, some individuals can also be designated as "groups" according to characteristics such as age, whether they smoke, whether they drink alcohol, and personal health status. "Normal GPRC5D content" in a population can be determined by measuring a sufficient number of individuals.

"Biological sample" means a quantity of material from a living or formerly living organism. Such substances include, but are not limited to, blood (eg, whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

The term "sequence identity" (also referred to as "sequence identity"), when referring to amino acid or nucleotide sequences, refers to the identity between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence). The amount of degree, usually expressed as a percentage. Typically, prior to calculating the percent identity between two amino acid or nucleotide sequences, the sequences are aligned and gaps, if any, introduced. If at a certain alignment position, the amino acid residues or bases in the two sequences are the same, the two sequences are considered to be identical or match at this position; if the amino acid residues or bases in the two sequences are different, they are considered to be inconsistent at this position or mismatch. In some algorithms, sequence identity is obtained by dividing the number of matching positions by the total number of positions in the alignment window. In other algorithms, the number of gaps and/or gap lengths are also taken into account. For the purposes of the present invention, the publicly available alignment software BLAST (available at ncbi.nlm.nih.gov) can be used to obtain an optimal sequence alignment and calculate two amino acid or nucleotide alignments by using default settings. Sequence identity between sequences.

GPRC5D binding peptide

Provided herein are GPRC5D binding peptides that specifically bind GPRC5D. The GPRC5D binding peptide binds the GPRC5D molecule with relatively high binding affinity. The GPRC5D can be a fusion protein linked to other polypeptides, such as GPRC5D-VLP, or a GPRC5D membrane protein expressed on the cell surface. For example, as described in the examples below, the binding ability of GPRC5D-binding peptides to GPRC5D can be measured by assay methods such as enzyme-linked immunosorbent assay (ELISA) and flow cytometry fluorescence sorting technique (FACS). In addition, it can also be determined by other protein interaction assay methods known in the art, for example, surface plasmon resonance (SPR) biolayer interferometry (BLI) technique.

In some embodiments, the GPRC5D binding peptide is an antibody molecule or an antigen-binding fragment thereof. Preferably, the GPRC5D-binding peptide is a single domain antibody.

As described in the examples herein, the GPRC5D-binding peptide provided herein comes from the screening of a human phage antibody library and is a fully human single domain antibody.

Single domain antibodies are antibodies that include only the variable region of the heavy chain, examples of which may include, but are not limited to, heavy chain antibodies, antibodies naturally lacking light chains, single domain antibodies derived from classical 4 chain antibodies, and engineered antibodies . Single domain antibodies may be derived from any species including, but not limited to, mouse, human, camel, llama, alpaca, vicuna, guanaco, shark, goat, rabbit, and/or bovine.

This paper provides the CDR sequences of three GPRC5D-binding peptides, which are respectively the sequences shown in SEQ ID NO: 1-3, 4-6, and 7-9.

On the basis of the CDR sequence of the GPRC5D binding peptide (single domain antibody) provided herein, those skilled in the art can construct various polypeptide constructs with GPRC5D binding ability, which include the use of framework regions (FR) and These CDR sequences are combined. These framework regions include native framework region sequences from human antibodies or animal (eg, mouse, rat, goat, camel, etc.) antibodies. These framework regions may also include framework region sequence variants produced by changes to the natural framework region sequences. A polypeptide construct specifically binding to GPRC5D can be easily obtained by combining the CDR sequences provided herein with different framework region sequences to form a heavy chain variable region, and testing its ability to bind to GPRC5D.

In some embodiments, the heavy chain variable region of the GPRC5D binding peptide (single domain antibody) provided herein comprises at least 90% sequence identity (e.g., at least 95%, at least 98%) to the sequence shown in SEQ ID NO: 10. %, at least 99% or even 100% sequence identity).

In some embodiments, the heavy chain variable region of the GPRC5D binding peptide (single domain antibody) provided herein comprises at least 90% sequence identity (e.g., at least 95%, at least 98%) to the sequence shown in SEQ ID NO: 11. %, at least 99% or even 100% sequence identity).

In some embodiments, the heavy chain variable region of the GPRC5D binding peptide (single domain antibody) provided herein comprises at least 90% sequence identity (e.g., at least 95%, at least 98%) to the sequence shown in SEQ ID NO: 12. %, at least 99% or even 100% sequence identity).

Those skilled in the art can understand that, on the basis of the specific sequences provided herein, a few amino acids can be replaced, deleted, added and verified or screened for the binding ability or biological activity of the resulting product to the corresponding antigen GPRC5D, thereby obtaining the present invention. Corresponding variants of the provided antibody molecules targeting GPRC5D should also be included within the scope of the present invention. For example, the antibody molecule or single domain antibody provided herein may have at least 1 and no more than 10, such as no more than 5, 4, 3, 2 or 1 amino acid in its full-length or variable region sequence or CDR sequence change.

It is contemplated that the GPRC5D binding peptides described herein may contain conservative amino acid substitutions. Conservative amino acid substitutions can generally be described as the substitution of one amino acid residue for another amino acid residue of similar chemical structure and have little or no substantial effect on the function, activity or other biological properties of the polypeptide. Conservative amino acid substitutions are well known in the art. Conservative substitutions can be, for example, the substitution of one amino acid in the following groups (a)-(e) by another amino acid within the same group: (a) small aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar positively charged residues: His, Arg and Lys; (d) large aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

The polypeptide construct may also include more than one GPRC5D-binding peptides, which are directly connected in series or connected in series through a linker sequence (linker). In some embodiments, the polypeptide construct includes two GPRC5D-binding peptides, the two GPRC5D-binding peptides being the same. In some embodiments, the polypeptide construct comprises two GPRC5D-binding peptides that differ in amino acid sequence by at least one amino acid. In some embodiments, the polypeptide construct includes at least one GPRC5D-binding peptide provided herein and at least one other GPRC5D-binding peptide. In some embodiments, the polypeptide construct includes two different GPRC5D-binding peptides provided herein. In some embodiments, the polypeptide construct includes three different GPRC5D-binding peptides provided herein.

The linker sequence can be a naturally occurring linker, a synthetic linker, or a combination of both. Particularly suitable linker sequences mainly comprise amino acid residues selected from the group consisting of glycine (Gly), serine (Ser), alanine (Ala) and threonine (Thr). For example, the linker may contain at least 75% (calculated based on the total number of residues present in the peptide linker), such as at least 80%, at least 85% or at least 90%, of amino acids selected from Gly, Ser, Ala and Thr Residues. Linkers may also consist of only Gly, Ser, Ala and/or Thr residues. In some embodiments, the linker contains 1-25 glycine residues, 5-20 glycine residues, 5-15 glycine residues, or 8-12 glycine residues. In some aspects, suitable peptide linkers typically contain at least 50% glycine residues, such as at least 75% glycine residues. In some embodiments, the peptide linker comprises only glycine residues. In some examples, the peptide linker comprises only glycine and serine residues, such as in the form (GS) _n , where n is, for example, an integer from 1-20.

Fusion proteins comprising GPRC5D-binding peptides

The above-mentioned polypeptide constructs comprising multiple GPRC5D-binding peptides belong to fusion proteins comprising GPRC5D-binding peptides. In addition, the GPRC5D binding peptide can also form a fusion protein with other polypeptides.

In some embodiments, a GPRC5D binding peptide can be linked to an Fc fragment to form a fusion protein. The Fc fragment can be located at the C-terminus and N-terminus of the GPRC5D binding peptide. Preferably, the Fc fragment may be located at the C-terminus of the GPRC5D binding peptide. The fusion protein formed by the GPRC5D-binding peptide and the Fc fragment has the ability to specifically bind GPRC5D, and at the same time has the effector function of the Fc fragment, such as mediating complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) , Mediate phagocytosis, etc. In addition, the fusion with the Fc fragment can increase the half-life of the GPRC5D-binding peptide in vivo, so as to increase the dosing interval when the GPRC5D-binding peptide is used as a therapeutic drug. Non-limiting fusion protein constructs containing GPRC5D-binding peptides may include, but are not limited to, the following formats: VHH1-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc, wherein VHH refers to GPRC5D-binding peptides (single domain antibodies), VHH1, VHH2 and VHH3 may be the same or different.

In some embodiments, a GPRC5D binding peptide can be linked to a protein tag to form a fusion protein. Protein tags can include purification tags and detectable tags. Purification tags include, but are not limited to, His6 tags, Flag tags, MBP tags, GST tags, SUMO) tags, and the like. The detectable label can be used to indicate the presence or content of the GPRC5D-binding peptide in the sample, or to track the location information of the GPRC5D-binding peptide in the body or cells of the subject. Examples of detectable labels include various enzymes useful in immunoassays, such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), etc.; fluorescent proteins, such as GFP. Due to the specific binding ability of the GPRC5D-binding peptide to GPRC5D, the amount of the GPRC5D-binding peptide can be determined by the amount of the detectable label linked to the GPRC5D-binding peptide, and then the content of GPRC5D in the sample can be determined.

In other embodiments, GPRC5D-binding peptides can be linked to cytokines or therapeutic proteins to form fusion proteins. In this case, the specific binding ability of GPRC5D-binding peptides to GPRC5D can be used to deliver cytokines or therapeutic proteins to specific tissues or cells (such as tumor tissues expressing GPRC5D) to achieve cytokine or therapeutic effects. The therapeutic effect of proteins.

Multispecific antibodies comprising GPRC5D-binding peptides

In some embodiments, provided herein are multispecific binding peptides comprising at least one GPRC5D-binding domain (or functional unit) and one or more additional binding domains. The one or more additional binding domains may bind to a second antigen or protein other than GPRC5D.

In some embodiments, provided herein are multispecific binding peptides comprising at least two GPRC5D-binding domains. The two binding domains binding to GPRC5D have differences in amino acid sequence, and respectively bind to different epitopes on GPRC5D.

In some embodiments, provided herein are multispecific binding peptides comprising at least two GPRC5D-binding domains and one or more additional binding domains. The two binding domains that bind GPRC5D differ in amino acid sequence and bind to different epitopes on GPRC5D respectively; the one or more additional binding domains may bind to a second antigen or protein other than GPRC5D .

In some embodiments, the GPRC5D-binding domain is a single domain antibody provided herein; the one or more additional binding domains can be a single domain antibody, single chain antibody, or other antigen-binding fragment.

In some embodiments, the second antigen is a tumor-associated antigen (TAA) or a tumor microenvironment-associated antigen (TMEAA). In some embodiments, the second antigen is an immunomodulatory antigen, wherein the antigen is associated with enhancing or inhibiting signaling pathways in immune cells. In some embodiments, the second antigen is a T cell surface molecule, such as a component of the T cell receptor complex, eg, CD3 (including gamma, delta, epsilon, zeta and eta chains).

Preferably, said multispecific binding peptide is a bispecific antibody molecule.

In some embodiments, the multispecific binding peptide also includes an Fc fragment. The presence of the Fc fragment facilitates multimerization of the binding domains and provides associated effector functions.

Immunoconjugates (or Antibody Conjugates) Including GPRC5D Binding Peptides

Provided herein are conjugates comprising at least one GPRC5D-binding peptide provided herein that specifically binds GPRC5D and one or more other functional moieties. The other moiety may be a chemical group, eg a therapeutic agent, such as a cytotoxic agent, or may be a tracer. In some examples, the moiety can be a targeting moiety, a small molecule drug (e.g., a non-polypeptide drug less than 500 Da), a toxin, a cytostatic agent, a cytotoxic agent, an immunosuppressant, a radioactive agent suitable for diagnostic purposes, Radioactive metal ions for therapeutic purposes, etc.

In some embodiments, the immunoconjugate is an antibody drug conjugate (ADC) comprising one or more GPRC5D binding peptides provided herein and a therapeutic agent that is cytotoxic, inhibits cell growth, or provides some therapeutic benefit . In some embodiments, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (such as an enzymatically active toxin or fragment thereof of bacterial, fungal, plant or animal origin), or a radioisotope (i.e., a radioconjugate ). In some examples, the antibody drug conjugates provided herein allow targeted delivery of drug moieties to tumors. In some instances, this can lead to targeted killing of tumor cells.

In some examples, therapeutic agents include, for example, daunomycin, doxorubicin, methotrexate, vindesine, maytansinoids, and the like. In some instances, therapeutic agents are intracellularly active. In some examples, an immunoconjugate that binds GPRC5D is internalized and the therapeutic agent has cellular protein synthesis activity, blocks nucleic acid synthesis activity, and causes cell growth arrest or death.

In some embodiments, an immunoconjugate comprises one or more GPRC5D-binding peptides provided herein and a tracer. The immunoconjugates can be used for research or diagnostic purposes, eg for detection of cancer in vivo. Tracers can directly or indirectly produce a detectable signal. For example, tracers may be radioactive isotopes such as ³ H, ¹⁴ C, ³² P, ³⁵ S, ¹²³ I; fluorescent (fluorophore) or chemiluminescent (chromophore) compounds such as fluorescent isothiocyanates, rhodamine or fluorescein; imaging agents; or metal ions. In some embodiments, the tracer is a radioactive atom for scintigraphic studies, such ^as99Tc ^or123I , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), For example ⁸⁹ Zr, ¹²³ I, ¹⁹ F, ¹³ C, ¹⁵ N, ¹⁷ O. ⁸⁹ Zr can complex with various metal chelators and bind to antibodies, for example for PET imaging.

Linkage of GPRC5D binding peptides to other functional moieties may be covalent or non-covalent. An example of non-covalent attachment may include via the Biotin-Avidin System. Examples of covalent linkages can include various chemical linkers, including peptide linkers, cleavable linkers, or non-cleavable linkers. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val -cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio base) pentanoate (“SPP”), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”) and N - Succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB").

In some embodiments, linkers may comprise amino acid residues. Exemplary amino acid linker components include dipeptides, tripeptides, tetrapeptides or pentapeptides. Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (afa-phe). Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues comprising amino acid linker components include naturally occurring residues as well as non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker components can be designed and optimized with respect to their selectivity for enzymatic cleavage by specific enzymes such as tumor-associated proteases, cathepsins B, C and D, plasmin proteases.

Conjugates of GPRC5D-binding peptides with cytotoxic agents can be prepared using a variety of bifunctional protein coupling reagents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP) , iminothiolane (IT), bifunctional derivatives of imide esters (such as dimethyl adipimide HCl), active esters (such as disuccinimidyl substrates), Aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), dinitrogen derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine ), diisocyanates (such as toluene 2,6-diisocyanate), and bisactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

Pharmaceutical compositions and methods of treatment comprising GPRC5D binding peptides

The GPRC5D-binding peptides disclosed herein, as well as fusion proteins comprising the GPRC5D-binding peptides, multispecific antibody molecules, and immunoconjugates (which can be formulated as pharmaceutical compositions together with pharmaceutically acceptable carriers) can be used for administration to subjects for cancer prevention or treatment. Amounts effective for this use will depend on the severity of the disease and the general state of the patient's own immune system. The dosing regimen will also vary with the disease state and the state of the subject, and will generally range from a single bolus or continuous infusion to multiple daily doses (eg, every 4-6 hours). A clinician skilled in the art can readily determine whether a subject is a candidate for such treatment, eg, by utilizing clinical tests, physical examination and the subject's family history.

In some embodiments, the GPRC5D-binding peptides disclosed herein and fusion proteins, multispecific antibody molecules, and immunoconjugates comprising the GPRC5D-binding peptides can be administered in combination with one or more other drugs (such as anti-tumor agents) medicine.

In some embodiments, the disease or condition treated is a tumor, preferably multiple myeloma. The GPRC5D-binding peptides disclosed herein (as well as fusion proteins, multispecific antibody molecules, and immunoconjugates comprising the GPRC5D-binding peptides) disclosed herein may be used in any suitable method or approach, optionally in combination with other anti-tumor agents. Routes of administration include, for example, oral, intravenous, intraperitoneal, subcutaneous or intramuscular administration.

The nucleic acid molecule encoding the GPRC5D-binding peptide provided herein, the expression vector comprising the nucleic acid molecule, and the host cells transfected with the nucleic acid molecule or the expression vector can also be used for the above-mentioned therapeutic purposes in various ways. For example, as for the expression vector, it can be introduced into the body of the subject by means of gene therapy known in the art to express the target protein or polypeptide (GPRC5D binding peptide), so as to achieve the purpose of treatment.

Assays, diagnostic kits or therapeutic kits comprising GPRC5D binding peptides

The GPRC5D-binding peptides provided herein, as well as other forms of molecules (such as fusion proteins or bispecific antibody molecules) comprising the GPRC5D-binding peptides, can specifically bind to GPRC5D in a sample. By detecting the amount of the formed GPRC5D-binding peptide-GPRC5D complex, or the amount of the GPRC5D-binding peptide in the formed GPRC5D-binding peptide-GPRC5D complex, the content (or presence) of GPRC5D in the sample can be conveniently determined.

As described above, for this purpose, the GPRC5D-binding peptides provided herein can be linked to various detection tags to facilitate detection by various means, including but not limited to bioluminescence, fluorescence, radiolabeling, enzymatic reaction. product production, etc. After detecting the GPRC5D content in the sample, comparing it with the normal GPRC5D content in the normal population can be used to determine the disease status or severity of the subject who provided the sample. By repeatedly detecting the change of GPRC5D content over time during the subject's treatment process, it can also be used to determine whether the treatment is effective, thereby providing a basis for the modification of the treatment plan.

The GPRC5D-binding peptides provided herein, as well as other forms of molecules comprising the GPRC5D-binding peptides, such as fusion proteins or bispecific antibody molecules, can be placed in containers to form detection, diagnostic or therapeutic kits. These containers may be in the form of cartridges, ampoules, vials, tubes, bags or other suitable containers known in the art. These containers can be made of plastic, glass, laminated paper, foil, or other materials suitable for holding medications. Instructions for use are provided with the container, if desired. The instructions may generally include information on how to use the GPRC5D-binding peptide or a composition comprising the GPRC5D-binding peptide for the treatment or prevention of tumors (e.g., multiple myeloma), for example may include a description of a therapeutic agent (e.g., a GPRC5D-binding peptide); Dosage regimens for the treatment or prevention of neoplasia (eg, multiple myeloma); precautions; warnings; indications; contraindications; adverse reactions; animal pharmacology; clinical studies; and/or reference materials. The instructions may be printed directly on the container (if present), or as a label affixed to the container, or as a separate paper, booklet, card or folded print provided in or with the container.

The GPRC5D-binding peptide (or GPRC5D-targeting antibody molecule) provided herein is a fully human single-domain antibody, and its binding affinity to GPRC5D is close to or better than that of the control antibody.

Research overview:

We used a large-capacity phage antibody library to screen fully human GPRC5D-specific antibodies, and used enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA) and flow cytometry fluorescence sorting technology (fluorescence activated cell sorting, FACS) Experiments were performed to evaluate the specificity of these antibodies at the phage level and protein level, and then FACS was used to measure the binding affinity of antibodies to cells, and these antibody molecules were constructed on the second-generation CAR lentiviral expression vectors, transfected into T cells, and passed in vitro The cell killing assay preliminarily evaluates the killing function of these antibody molecules. Finally, we obtained three fully human antibody clones with good specificity and strong killing function.

We used different antibody libraries, after recombinant GPRC5D protein panning and GPRC5D protein/cell (CHO-K1-GPRC5D) panning, a total of 768 monoclonals were selected for enzyme-linked immunosorbent assay (ELISA) and flow cytometry ( FACS) detection preliminary screening, in which 216 clones specifically combined with GPRC5D-VLP protein and GPRC5D expression-positive cell CHO-K1-GPRC5D, but did not bind to the control protein VLP and GPRC5D-expressed negative cell CHO-K1. After sequencing, 35 different monoclonal sequences were obtained. Subsequently, we performed flow cytometric analysis (FACS) identification of these 35 antibodies against multiple GPRC5D-positive (CHO-K1-GPRC5D, MM1S) and negative cell lines (CHO-K1, Jurkat) and identified them with multiple non-related antigens ( VLP, BAFFR-his-Bio, IL10-Bio, SA) and GPRC5D protein (GPRC5D-VLP) were identified by enzyme-linked immunosorbent assay (ELISA), among which 3 clones were expressed on various cell lines and various protein antigens Good binding and specificity. The acquisition of these clones laid the foundation for the subsequent development of fully human GPRC5D CAR-T products or antibody drugs.

The present invention will be described in detail below in conjunction with specific embodiments.

Example 1. Enrichment of specific antibody clones targeting GPRC5D protein from phage antibody library by affinity panning

Use appropriate negative panning and positive panning strategies to enrich the specific antibody clones we need from the phage antibody library.

Construction of phage antibody library

The phage antibody libraries we constructed include natural libraries, semi-synthetic libraries and single-domain libraries. Semi-synthetic phage antibody library, used together with natural library, to solve the problem that natural library may lack GPRC5D high-affinity antibody clones. The single-domain phage antibody library is an antibody library composed only of the variable region amino acids of heavy chain antibodies. Its molecular weight is only 12-15kDa, but it has similar or higher specificity and affinity than traditional antibodies. In addition, single domain antibodies have attracted much attention because of their stable physical and chemical properties, high affinity, easy recombinant expression and preparation, and easy combination with other target or epitope antibodies.

GPRC5D protein panning

Using GPRC5D-VLP as the positive panning protein and VLP protein as the negative panning protein, multiple rounds of panning were performed to obtain a phage pool enriched for the target antibody clone. The experimental steps are briefly described as follows:

1) Coating antigen: Dilute the antigen GPRC5D-VLP to 10 μg/mL with clean coating buffer (PBS), add 100 μL working solution to each well, panning 6 wells per panning, and bind overnight at 4°C;

Dilute the antigenic VLP protein with clean coating buffer (PBS) to 10 μg/mL, add 100 μL of working solution to each well, panning 6 wells per panning, and bind overnight at 4°C; negative panning antigens are labeled as negative plates, positive panning Antigen is a positive plate;

2) Blocking: remove the antigen upside down, and pat off the residual liquid in the well with absorbent paper, add 250 μL of 3% BSA-PBS to block, and block at room temperature for 2 hours;

3) adding a phage library (containing 5× ¹⁰ phage particles) and incubating with the control antigen VLP, so as to deduct phage antibody clones that do not specifically bind to VLP;

4) After incubation, the supernatant was transferred to a positive plate bound to the target antigen GPRC5D-VLP, and the incubation was continued to allow the phage to bind to the target antigen;

5) washing the magnetic beads with washing solution to wash away unbound phage;

6) Use the eluent to elute the positive phage from the target antigen, and add neutralizing solution to neutralize;

7) Re-infect the host strain XL1-blue with the eluted phage, and amplify the recovered phage. Leave a small amount of sample for gradient dilution, infect the host bacteria, apply Amp resistance plates, and calculate the number of recovered phages;

8) Repeat steps 1) to 6), usually three rounds of panning are required until the recovery rate of phages (number of eluted phages/number of input phages) is significantly increased.

The enriched phage pool can be used for subsequent monoclonal selection and ELISA/FACS screening.

Main materials and reagents:

Fully human phage antibody library, including natural library, semi-synthetic library and single domain library;

Helper phage KO7, Thermo/Invitrogen, 18311019;

Human GPRC5D Protein-VLP, Kactus, GPR-HM05P;

VLP protein, Kactus, order

High binding ELSIA plate, Costar, #3590

Blocking solution: PBS+3%BSA

Rinse solution: PBS+0.1% Tween20

Eluent: 0.2M Glycine, pH2.2

Neutralizing solution: 1M Tris, pH9.1

Experimental results:

Using different antibody libraries, through 3 rounds of protein panning, a significant increase in the recovery rate was observed in each panning (Table 1), proving that antibody clones were effectively enriched.

Table 1 Results of protein panning experiments

It can be seen that after three rounds of panning, different antibody libraries were enriched (the recovery rate of the third round was significantly higher than that of the previous round). However, the specific binding clones screened by this panning strategy were less, and in the subsequent FACS experiments, the binding to CHO-K1-GPRC5D cells with high expression of GPRC5D antigen was poor, that is, they could not recognize the natural GPRC5D antigen on the cell surface . Therefore, in subsequent experiments, we used the method of protein and cell alternate panning to enrich specific antibody clones that can simultaneously bind to GPRC5D protein and the natural state of GPRC5D on the cell surface from the phage antibody library. Table 2 shows the results of joint panning using recombinant GPRC5D protein and CHO-K1-GPRC5D/CHO-K1 cell line. Judging from the recovery rate, all 4 pannings were enriched and could be used to select single clones in the next step.

Table 2 Results of protein+cell panning experiments

Example 2. Screening of specific clones from enriched phage pools using ELISA and FACS

Purpose and principle: The phage pool enriched by the affinity panning step contains phage antibodies of various properties: specific clones, non-specific clones, and negative clones. In order to obtain specific clones, we need to isolate single clones, package them into monoclonal phages, and conduct primary screening on a large number of single clones by ELISA) and FACS, and select the positive cell line CHO- Monoclonal K1-GPRC5D. The specific monoclonal is further determined by DNA sequencing to determine the unique antibody sequence contained therein.

In the primary screening by ELISA, those that only bind to GPRC5D-VLP but not to the control antigen VLP were identified as specific clones. The FACS primary screening was carried out using the positive cell line CHO-K1-GPRC5D with high expression of GPRC5D and the cell line CHO-K1 negative for GPRC5D, and those that only combined with CHO-K1-GPRC5D and not with CHO-K1 cells were identified as specific clones. Through ELISA and FACS, we can obtain candidate antibodies that can bind to recombinantly expressed GPRC5D protein and recognize the natural state GPRC5D molecule on the cell surface for subsequent further screening.

Brief steps of ELISA primary screening experiment:

1) Cultivate and package monoclonal phage in a deep-well 96-well plate;

2) Dilute GPRC5D-VLP to 10 μg/mL with PBS, add 100 μL/well to a high-binding microtiter plate, and coat overnight at 4°C;

3) Discard the coating solution, add 250 μL of blocking solution to each well, and block at room temperature for 2 hours;

4) Wash the plate twice with 250 μL of washing solution;

5) Add 100 μL of the phage supernatant cultured in step 1) to the wells coated with the target antigen, and combine at room temperature for 2 hours;

6) Wash the plate 4 times with 250 μL of washing solution;

7) Add mouse anti M13 primary antibody diluted 1:2000, 100 μL/well, incubate at room temperature for 45 minutes;

8) Wash the plate 4 times with 250 μL of washing solution;

9) Add 1:2000 diluted HRP Donkey anti-mouse IgG, 100 μL/well, and incubate at room temperature for 45 minutes;

10) Wash the plate 6 times with 250 μL rinse solution;

11) Add 100 μL TMB chromogenic substrate, and develop color for 10 minutes;

12) Add 100 μL of 2M H ₂ SO ₄ to terminate the reaction, and read the result on a microplate reader.

Brief steps of FACS primary screening experiment:

1) Cultivate and package monoclonal phage in a deep-well 96-well plate;

2) Wash CHO-K1-GPRC5D and CHO-K1 cells twice with PBS, resuspend in PBS to a concentration of 1x10 ⁷ /mL, and dispense 50 μL into 96-well deep-well plates;

3) Add 50 μL of packaged monoclonal phage to each well, mix well, and combine at 4°C for 2 hours;

4) Wash twice with 200 μL PBS;

5) Add mouse anti M13 primary antibody diluted 1:2000, 100 μL/well, mix well by pipetting, and incubate at room temperature for 45 minutes;

6) Wash twice with 200 μL PBS;

7) Add 1:300 diluted FITC horse anti mouse-IgG (H+L), 100 μL/well, mix by pipetting, and incubate at room temperature for 45 minutes;

8) Wash twice with 200 μL PBS; finally resuspend the cells with 200 μL PBS;

9) Detect the fluorescence intensity of the FITC channel of the sample on the flow cytometer, and analyze the results.

Main materials and reagents:

Helper phage KO7, Thermo/Invitrogen, 18311019

Streptavidin, Pierce, 21125

Human GPRC5D Protein-VLP, Kactus, GPR-HM05P;

Biotinylated Human IL-10Protein, Kactus, IL1-HM410B;

Biotinylated Recombinant human BAFFR Protein, Kactus, BAF-HM40RB;

High binding ELSIA plate,Costar,#3590

Corning 96 Well Clear Round Bottom TC-Treated Microplate,Costar,#3799

Blocking solution: PBS+3%BSA

Rinse solution: PBS+0.1% Tween20

Soluble one-component TMB substrate solution, Tiangen, PA-107-02

Anti-M13Bacteriophage Coat Protein g8p antibody,abcam,ab9225

HRP Goat anti-mouse IgG (minimal x-reactivity) Antibody, Biolegend, 405306

HRP Donkey anti-human IgG (minimal x-reactivity) Antibody, Biolegend, 410902

HRP Conjugated Anti His-Tag Mouse Monoclonal Antibody, CWBIO, CW0285

FITC horse anti mouse-IgG(H+L),Vector,FI2000

H_GPRC5D CHO-K1 Cell Line Clone, Shanghai Jiman, GM-C03857

Human GPRC5D PE-conjugated Antibody, RD, FAB6300RP

Fluorescein (FITC) AffiniPure Goat Anti-Human IgG, Fcγfragment specific, Jackson

ImmunoReseach, 109-095-190

Experimental results:

Monoclonals were randomly selected from the enriched phage antibody pool, packaged into phages, and the binding of monoclonal phages to GPRC5D-VLP protein and control protein VLP was detected by phage ELISA to find GPRC5D-specific phage antibody clones. The ELISA results of some clones are shown in Figure 1. It can be seen from the figure that clones A1, A2, A4, A7 and A8 bind strongly to the target antigen GPRC5D (GPRC5D-VLP), and do not bind to the control antigen VLP, showing good specificity. Clones A3, A5 and A6 combined with both the target antigen and the control antigen, and did not meet the requirements for specific binding. Negative phage control is a negative control phage antibody clone, which does not bind to the target antigen or control antigen. Anti-M13 phage mouse/anti-mouse HRP Ab is a negative antibody control with only primary and secondary antibodies added, and anti-mouse HRP Ab is Anti-human IgG HRP Ab is a negative antibody control with only secondary antibody added, which does not bind to the target antigen and control antigen; anti-his tag HRP Ab is a positive antibody control for detecting antigen tags, Combination with his-tagged antigen indicates that the coated antigen has been bound to the plate. Positive Benchamrk1 is a positive antibody to the target antigen, which binds to the target antigen and does not bind to the control antigen.

The results of FACS primary screening of antibody clones corresponding to ELISA are shown in Figure 2. Fig. 2A is the result after staining of CHO-K1-GPRC5D cell line by commercialized flow cytometry antibody Human GPRC5D PE-conjugated Antibody and Positive benchmark1 (variable region sequence comes from GC5B596 of Chinese patent application publication CN 109715667 A), the result shows that All cells can bind to GPRC5D antibody, indicating that CHO-K1-GPRC5D cells are positive for GPRC5D expression. The A1, A2, A3, A4, A5, A6 and A7 clones in Figure 2B bind to CHO-K1-GPRC5D, but do not bind to CHO -K1 cell is a specific clone; A8 clone does not combine with the two kinds of cells, it is a negative clone; Negative phage control is a negative control phage antibody clone, which does not bind to the target antigen and the control antigen, Positive Benchamrk1 Ab (GC5B596) is The positive antibody of the target antigen binds to the highly expressed cell line CHO-K1-GPRC5D, but does not bind to the control negative cell CHO-K1.

Through ELISA detection and FACS primary screening, we obtained a total of 216 ELISA and FACS double-positive clones with good specificity, and then we sequenced the 216 double-positive clones with good specificity, and obtained 35 different clones after sequencing. Then the 35 single clones with different sequences were further tested for the binding specificity of the candidate clones through FACS identification of multiple cell lines and ELISA identification of various antigens. In the following examples, three of the single domain antibody clones (clone

numbers

18, 39 and 41) were selected for experiments, and their HCDR sequences, heavy chain variable region (VH) sequences and VH encoding nucleic acid sequences are as follows.

Example 3. Further identification of monoclonal specificity at the phage level by ELISA and FACS

The purpose and principle of the experiment: the antibody used for treatment must have very good target specificity, and only bind the target antigen, not any unrelated antigen; on the other hand, the amino acid sequence of the same antigen on different cell lines will be different (Isomers or mutants) or binding ligands are different, and it is also necessary to investigate whether our antibodies can bind to cells positive for various target proteins. In order to further analyze the specificity and universality of these monoclonals and find the best candidate clones, we further evaluated the specificity of the primary screening clones by enzyme-linked immunoassay (ELISA) and flow cytometry.

Further identification of monoclonal specificity by ELISA using multiple unrelated antigens

In this experiment, we used the target antigen GPRC5D-VLP antigen and various GPRC5D-unrelated antigens to react with these monoclonal phage antibodies, and analyzed whether these clones had any non-specific binding to other GPRC5D-unrelated antigens. Through this experiment, we obtained several clones with excellent specificity.

Experimental method: the same as the ELISA primary screening;

Main samples and reagents:

The rest of the reagents are the same as the ELISA primary screening.

Experimental results:

Antibodies used for therapy must have very good target specificity. In order to further analyze the specificity of these monoclonal antibodies, we identified multiple clones obtained in Example 2 on various antigens using enzyme-linked immunosorbent assay (ELISA). The results are shown in Figure 3. Negative phage control is a negative control phage antibody clone that does not bind to the target antigen or control antigen. Anti-M13 phage mouse/anti-mouse HRP Ab is a negative antibody control that only adds primary and secondary antibodies , anti-mouse HRP Ab is the negative antibody control with secondary antibody only, they do not bind to the target antigen and the control antigen, Positive Benchmark1 Ab (GC5B596) is the positive antibody control of the target antigen (GPRC5D-VLP), it binds to the target antigen , does not bind to the control antigen. Anti-human IgG HRP Ab is a negative antibody control with only secondary antibody added, and anti-his HRP Ab is a positive antibody control for detecting antigen tags, which binds to the his-tagged antigen, indicating that the coated antigen has been bound to the plate.

Clones

18, 39, and 41 all bound to the GPRC5D antigen, but none of the four non-related antigens, indicating that

clones

18, 39, and 41 could bind to the GPRC5D antigen with good specificity.

Further identification of monoclonal specificity by FACS at the phage level using multiple cell lines

In this experiment, we used a variety of GPRC5D-positive cell lines and a variety of GPRC5D-negative cell lines to react with these monoclonal phage antibodies to analyze whether these clones can bind to GPRC5D antigens on different cell lines and whether they are compatible with other Cell lines that do not express GPRC5D have any non-specific binding. Through this experiment, we obtained several clones with excellent specificity.

Experimental method: the same as the FACS primary screening;

Main samples and reagents:

CHO-K1-GPRC5D cell line, GPRC5D positive cell line;

MM1S cell line, GPRC5D positive cell line;

CHO-K1 cell line, GPRC5D negative cell line;

Jurkat cell line, GPRC5D negative cell line;

The rest of the reagents were the same as the FACS primary screening.

Experimental results:

Antibodies used for therapy must have very good target specificity. In order to further analyze the specificity of these monoclonal antibodies, we identified the only clone obtained in Example 2 on more antigens and cell lines using ELISA and flow cytometry. The results are shown in Figure 4, Negative phage Control is a negative control phage antibody clone.

Clones

18, 39, and 41 combined with the two GPRC5D-positive cell lines CHO-K1-GPRC5D and MM1S, but did not bind with the two GPRC5D-negative cell lines CHO-K1 and Jurkat, with good specificity; Positive Benchamrk1 Ab (GC5B596) was The positive antibody of the target antigen binds to the highly expressed cell line CHO-K1-GPRC5D, but does not bind to the control negative cell CHO-K1; combined.

Example 4. Further determination of the binding specificity of the monoclonal by ELISA and FACS at the protein level

Experiment purpose and principle

Specific antibody clones targeting the GPRC5D antigen were enriched from the phage antibody library by affinity panning and screened and identified to obtain clones that bind to the GPRC5D antigen, but the antibody molecules expressed in the prokaryotic system were converted to those expressed in the eukaryotic system After the IgG antibody molecule, its binding ability and specificity need to be further confirmed. To this end, we prepared the IgG expression plasmids of these clones and expressed them by transiently transfecting CHOS cells, and Protein A was purified to antibodies. Then, the binding specificity of the monoclonals was further determined by ELISA and FACS.

Further confirm the binding specificity of the monoclonal at the antibody level by ELISA at the protein level

Main samples and reagents:

Same as ELISA reagents at phage level;

Experimental results:

In order to further analyze whether these monoclonal antibodies still maintain the binding ability and specificity of the original antibodies after being expressed as IgG antibodies in the eukaryotic system, we expressed the 3 clones obtained in Example 3 by CHOS, and purified Protein A to Antibodies in IgG form were identified on various antigens using enzyme-linked immunosorbent assay (ELISA). The results are shown in Figure 5. Positive Benchmark1 Ab is the positive antibody control of the target antigen (GPRC5D-VLP), which binds to the target antigen and does not bind to the control antigen. Anti-human IgG HRP Ab is a negative antibody control with only secondary antibody added, and anti-his HRP Ab is a positive antibody control for detecting antigen tags, which binds to the his-tagged antigen, indicating that the coated antigen has been bound to the plate.

Clones

clones

18, 39, and 41 could bind to the GPRC5D antigen with good specificity.

Further confirm the binding specificity of the monoclonal at the antibody level by FACS at the protein level

Main samples and reagents:

ImmunoReseach, 109-095-190

Experimental results:

In order to further analyze whether these monoclonal antibodies still maintain the binding ability and specificity of the original antibodies after being expressed as IgG antibodies in the eukaryotic system, we expressed the 3 clones obtained in Example 3 by CHOS, and purified Protein A to Antibodies in the IgG form were identified on more antigens and cell lines using ELISA and flow cytometry. The results are shown in Figure 6,

clones

18, 39 and 41 combined with the two GPRC5D-positive cell lines CHO-K1-GPRC5D and MM1S, and did not bind with the two GPRC5D-negative cell lines CHO-K1 and Jurkat, with good specificity; Positive Benchamrk1 Ab (GC5B596) is a positive antibody to the target antigen, which binds to the highly expressed cell line CHO-K1-GPRC5D, but does not bind to the control negative cell CHO-K1; FITC anti-human IgG Ab is a negative control that only adds secondary antibodies, Does not bind to either cell line.

Example 5. Detecting the Binding Ability of Monoclonal Antibodies to Highly Expressed Cell Lines by FACS

The affinity between GPRC5D antibody molecules and antigens may have an important impact on the killing effect and duration of CAR-T or antibody drugs in patients. Because it is difficult to obtain purified antigens for the target GPRC5D, we used the FACS binding method to analyze the half effective concentration (Ec50) of antibody molecules, which provided important information for the research and development process.

Brief steps of FACS binding experiment:

1) Preparation of different concentrations of antibodies: use PBS to dilute the anti-GPRC5D IgG from 300nM to 5-fold sequentially, and dilute to 0.00384nM for a total of 8 concentrations to prepare for the binding ability of the antibody to CHO-K1-GPRC5D cells;

2) Wash CHO-K1-GPRC5D cells twice with PBS, resuspend in PBS to a concentration of 1x10 ⁷ /mL, and dispense 50 μL into 96-well deep-well plates;

3) Add 50 μL of diluted antibody to each well, mix well, and combine at 4°C for 2 hours;

4) Wash twice with 200 μL PBS;

5) Add 1:300 diluted Alexa

647 AffiniPure Goat Anti-Human IgG, 100 μL/well, after mixing by pipetting, incubate at room temperature for 45 minutes;

6) Wash twice with 200 μL PBS; finally resuspend the cells with 200 μL PBS;

7) Detect sample Alexa on flow cytometer

Fluorescence intensity of 647 channels;

8) Binding constants were analyzed by using Graphpad Prism software.

Main samples and reagents:

CHO-K1-GPRC5D cell line, GPRC5D positive cell line;

Alexa

647 AffiniPure Goat Anti-Human IgG, Fcγfragment specific, Jackson ImmunoReseach, 109-605-008

Experimental results

Affinity refers to the binding strength of a single molecule to its ligand. Usually, FACS can be used to detect the binding ability to positive cell lines to evaluate the strength of the interaction between two molecules and rank them. The smaller the Ec50 value, the greater the affinity of the antibody for its target. As shown in Table 3 and Figure 7, Benchmark1, clone 18, clone 39 and clone 41 can all bind to the GPRC5D overexpression cell line CHO-K1-GPRC5D cells, and the affinity of clone 18 is slightly higher than that of Benchamrk1, higher than that of clone 39 and clone 41.

Table 3 Affinity determination results

抗体Antibody	Ec50(nM)Ec50(nM)	TOP(MFI)TOP(MFI)
克隆18 Clone 18	1.581.58	34623462
克隆39 Clone 39	6.936.93	27952795
克隆41 Clone 41	41.0241.02	36903690
Benchmark 1 Benchmark 1	6.726.72	25252525

references:

1.Voisinne G,Gonzalez de Peredo A,Roncagalli R.GPRC5D,an Undercover Regulator of TCR Signaling.Front Immunol.2018;9:2900.Published 2018 Dec 7.doi:10.3389/fimmu.2018.02900.

2. Lancman G, Sastow DL, Cho HJ, Jagannath S, Madduri D, Parekh SS, Richard S, Richter J, Sanchez L, Chari A. Bispecific Antibodies in Multiple Myeloma: Present and Future. Blood Cancer Discov. 2021 Aug 17; 2(5):423-433.doi:10.1158/2643-3230.BCD-21-0028.PMID:34661161;PMCID:PMC8510808.

3. Smith EL, Harrington K, Staehr M, Masakayan R, Jones J, Long TJ, Ng KY, Ghoddusi M, Purdon TJ, Wang X, Do T, Pham MT, Brown JM, De Larrea CF, Olson E, Peguero E , Wang P, LIU H, XU Y, Garrett-Thomson SC, Almo SC, Wendel Hg, RIVIERE I, LIU C, SATHER B, Brentjens RJ.GPRC5D IS A TARGET for the Immunotherapy of Multiple Myeloma with Rationally designed car t cells. Sci Transl Med.2019Mar 27;11(485):eaau7746.doi:10.1126/scitranslmed.aau7746.PMID:30918115;PMCID:PMC7508042.

4. Hosny M, Verkleij CPM, van der Schans J, Frerichs KA, Mutis T, Zweegman S, van de Donk NWCJ. Current State of the Art and Prospects of T Cell-Redirecting Bispecific Antibodies in Multiple Myeloma. J Clin Med .2021Oct 6;10(19):4593.doi:10.3390/jcm10194593.PMID:34640611;PMCID:PMC8509238.

5. Gagelmann N, Riecken K, Wolschke C, Berger C, Ayuk FA, Fehse B,

N.Development of CAR-T cell therapies for multiple myeloma.Leukemia.2020 Sep;34(9):2317-2332.doi:10.1038/s41375-020-0930-x.Epub 2020Jun 22.PMID:32572190.

6. Pillarisetti K, Edavettal S,

M, Li Y, Tornetta M, Babich A, Majewski N, Husovsky M, Reeves D, Walsh E, Chin D, Luistro L, Joseph J, Chu G, Packman K, Shetty S, Elsayed Y, Attar R, Gaudet FA T -cell-redirecting bispecific G-protein-coupled receptor class 5member D x CD3 antibody to treat multiple myeloma.Blood.2020 Apr 9;135(15):1232-1243.doi:10.1182/blood.2019003342.PMID:320405 49;PMCID :PMC7146017.

7. de Larrea CF, Staehr M, Lopez AV, Ng KY, Chen Y, Godfrey WD, Purdon TJ, Ponomarev V, Wendel HG, Brentjens RJ, Smith EL. Defining an Optimal Dual-Targeted CAR T-cell Therapy Approach Simultaneously Tar getting BCMA and GPRC5D to Prevent BCMA Escape-Driven Relapse in Multiple Myeloma.Blood Cancer Discov.2020Sep;1(2):146-154.doi:10.1158/2643-3230.bcd-20-0020.PMID:33089218; PMCID:PMC7575057 .

Claims

GPRC5D binding peptide, including the heavy chain variable region of the antibody molecule, HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are selected from one of the following combinations:

1) HCDR1 is GGSFSGYY (SEQ ID NO: 1);

HCDR2 is INHSGST (SEQ ID NO: 2);

The sequence of HCDR3 is ARARRYGGRTRFDP (SEQ ID NO: 3);

2) HCDR1 is GFIFSSYG (SEQ ID NO: 4);

The sequence of HCDR2 is ISSSGDYT (SEQ ID NO: 5);

The sequence of HCDR3 is ARMSFRRYDH (SEQ ID NO: 6); and

3) HCDR1 is GFSFSGYI (SEQ ID NO: 7);

The sequence of HCDR2 is TSSSGTET (SEQ ID NO: 8);

The sequence of HCDR3 is ARYYSKYGRSYHVDS (SEQ ID NO: 9).
The GPRC5D-binding peptide of claim 1, wherein the GPRC5D-binding peptide is an antibody molecule or an antigen-binding fragment thereof.
The GPRC5D binding peptide as claimed in claim 1 or 2, wherein said heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10, 11 or 12; or said heavy chain variable region comprises the same sequence as SEQ ID NO : The sequence shown in 10, 11 or 12 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity and can specifically Sexually binds GPRC5D.
The GPRC5D-binding peptide according to any one of claims 1-3, wherein the GPRC5D-binding peptide is a single domain antibody molecule.
The GPRC5D-binding peptide according to any one of claims 1-4, wherein the GPRC5D-binding peptide is a fully human antibody molecule.
The GPRC5D-binding peptide according to any one of claims 1-5, whose ability to bind to cells expressing GPRC5D on the surface has an EC50 value detected by FACS not higher than 10nM, 9nM, 8nM, 7nM, 6nM, or 5nM.
A fusion protein comprising at least one GPRC5D binding peptide according to any one of claims 1-6.
The fusion protein according to claim 7, comprising at least two GPRC5D binding peptides according to any one of claims 1-6.
The fusion protein according to any one of claims 7 or 8, wherein the HCDR1, HCDR2 and HCDR3 of the two GPRC5D-binding peptides are respectively selected from the different combinations listed in claim 1.
The fusion protein according to any one of claims 7-9, wherein:

One of the two GPRC5D binding peptides comprises the amino acid sequence shown in SEQ ID NO: 10, or comprises at least 90%, 91%, 92%, 93%, 94%, 95% of the sequence shown in SEQ ID NO: 10 %, 96%, 97%, 98%, or 99% sequence identity amino acid sequence and can specifically bind GPRC5D, the other comprises the amino acid sequence shown in SEQ ID NO: 11, or includes the amino acid sequence shown in SEQ ID NO: 11 An amino acid sequence showing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity and capable of specifically binding to GPRC5D;

One of the two GPRC5D binding peptides comprises the amino acid sequence shown in SEQ ID NO: 10, or comprises at least 90%, 91%, 92%, 93%, 94%, 95% of the sequence shown in SEQ ID NO: 10 %, 96%, 97%, 98%, or 99% sequence identity amino acid sequence and can specifically bind GPRC5D, the other includes the amino acid sequence shown in SEQ ID NO: 12, or includes the amino acid sequence shown in SEQ ID NO: 12 An amino acid sequence showing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity and capable of specifically binding GPRC5D; or

One of the two GPRC5D binding peptides includes the amino acid sequence shown in SEQ ID NO: 11, or includes at least 90%, 91%, 92%, 93%, 94%, 95% of the sequence shown in SEQ ID NO: 11 %, 96%, 97%, 98%, or 99% sequence identity amino acid sequence and can specifically bind GPRC5D, the other includes the amino acid sequence shown in SEQ ID NO: 12, or includes the amino acid sequence shown in SEQ ID NO: 12 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity and capable of specifically binding to GPRC5D is indicated.
The fusion protein according to any one of claims 7-10, wherein the fusion protein comprises at least three GPRC5D-binding peptides, wherein the HCDR1, HCDR2 and HCDR3 of the three GPRC5D-binding peptides are claims The three combinations listed in 1.
The fusion protein according to any one of claims 7-11, wherein the first of the three GPRC5D binding peptides of the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 10, or comprises the same sequence as SEQ ID NO: The sequence shown in 10 has an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity and can specifically bind GPRC5D the second comprises the amino acid sequence shown in SEQ ID NO: 11, or comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity amino acid sequence and can specifically bind to GPRC5D; and the third includes the amino acid sequence shown in SEQ ID NO: 12, or includes a An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity and capable of specifically binding GPRC5D.
The fusion protein according to any one of claims 7-12, wherein the fusion protein further comprises an Fc fragment.
The fusion protein according to any one of claims 7-13, wherein the fusion protein further comprises another binding peptide capable of specifically binding an antigen different from GPRC5D.
The fusion protein according to any one of claims 7-14, wherein the other binding peptide is a single domain antibody or a single chain antibody.
The fusion protein according to any one of claims 7-15, wherein the fusion protein further comprises a detectable label or a purification label.
The fusion protein according to any one of claims 7-16, wherein the detectable label has enzymatic activity.
A nucleic acid molecule encoding the GPRC5D-binding peptide of any one of claims 1-6 or the fusion protein of any one of claims 7-17.
The nucleic acid molecule of claim 18, wherein said nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 13, 14 or 15.
An expression vector comprising the nucleic acid molecule of claim 18 or 19.
A host cell comprising the nucleic acid molecule of claim 18 or 19 or the expression vector of claim 20.
A multispecific antibody molecule comprising at least a first functional part and a second functional part, wherein the first functional part comprises the GPRC5D binding peptide described in any one of claims 1-6; the second functional part has the same function as the first functional part Partially different binding specificities.
The multispecific antibody molecule of claim 22, wherein the second functional portion has binding specificity for immune cells.
The multispecific antibody molecule of claim 22 or 23, wherein the second functional portion has binding specificity for T cells.
The multispecific antibody molecule of any one of claims 22-24, wherein the second functional portion has binding specificity for CD3.
An immunoconjugate comprising the GPRC5D-binding peptide of any one of claims 1-6 linked to a therapeutic agent.
The immunoconjugate of claim 26, wherein the therapeutic agent is a drug.
The immunoconjugate of claim 26 or 27, wherein the therapeutic agent is a cytotoxin.
The immunoconjugate of any one of claims 26-28, wherein the therapeutic agent is a radioisotope.
Pharmaceutical compositions, comprising:

1) the GPRC5D binding peptide described in any one of claims 1-6;

The fusion protein according to any one of claims 7-17;

The multispecific antibody molecule of any one of claims 22-25; or

the immunoconjugate of any one of claims 26-29; and

2) A pharmaceutically acceptable carrier.
The GPRC5D-binding peptide of any one of claims 1-6, the fusion protein of any one of claims 7-17, the multispecific antibody molecule of any one of claims 22-25, or the multispecific antibody molecule of any one of claims 26- Use of the immunoconjugate described in any one of 29 in the preparation of a medicament for treating GPRC5D-related diseases.
The use according to claim 31, wherein the GPRC5D-associated disease is cancer or autoimmune disease, the cancer is preferably a plasma cell malignant tumor disease, such as multiple myeloma, or a B cell malignant disease, such as Hodgkin's lymphoma or non-Hodgkin's lymphoma.
A method for treating GPRC5D-related diseases, comprising an effective amount of the GPRC5D binding peptide described in any one of claims 1-7, the fusion protein described in any one of claims 7-17, the fusion protein described in any one of claims 22-25 The multispecific antibody molecule described above, the immunoconjugate described in any one of claims 26-29 or the pharmaceutical composition described in claim 30 is administered to a subject in need.
The method of claim 33, wherein the GPRC5D-associated disorder is cancer or an autoimmune disease.
The method of claim 34, wherein the cancer is a plasma cell malignancy or a B cell malignancy.
The method of claim 35, wherein said plasma cell malignancy disease is multiple myeloma and said B cell malignancy disease is Hodgkin's lymphoma or non-Hodgkin's lymphoma.
A method for detecting the content of GPRC5D in a biological sample, comprising:

1) making the biological sample with the GPRC5D binding peptide described in any one of claims 1-6, the fusion protein described in any one of claims 7-17 or the multispecific protein described in any one of claims 22-25 contact with the antibody molecule to form a binding complex with said GPRC5D;

2) Determining the content of GPRC5D in the biological sample based on the amount of the binding complex generated in step 1).
A method for detecting the content of GPRC5D in a biological sample, comprising:

1) coupling the GPRC5D-binding peptide of any one of claims 1-6 to a detectable label;

2) contacting said GPRC5D-binding peptide bearing said detectable label with said biological sample; and

3) determining the content of GPRC5D in the biological sample by detecting the amount of the detectable label.
A detection kit comprising the GPRC5D binding peptide according to any one of claims 1-6, the fusion protein according to any one of claims 7-17 or the multispecific antibody molecule according to any one of claims 22-25 .
A pharmaceutical kit, comprising the GPRC5D binding peptide according to any one of claims 1-6, the fusion protein according to any one of claims 7-17 or the multispecific antibody molecule according to any one of claims 22-25 .
A method of identifying a GPRC5D-associated disorder in a subject, comprising:

1) using the detection kit of claim 39 to determine the content of GPRC5D in the biological sample from the subject; and

2) Compared with the normal GPRC5D content in the population to determine whether the GPRC5D-related disease exists or its status.
The method of claim 41, wherein the GPRC5D-associated disorder is cancer or an autoimmune disease.
The method of claim 42, wherein the cancer is a plasma cell malignancy or a B cell malignancy.
The method of claim 43, wherein said plasma cell malignancy disease is multiple myeloma and said B cell malignancy disease is Hodgkin's lymphoma or non-Hodgkin's lymphoma.
A method for determining the therapeutic effect of a drug for treating a GPRC5D-related disorder in a subject, comprising:

1) using the method described in any one of claims 42-44 to identify the GPRC5D-related disease state of the subject;

2) treating the subject with the drug; and

3) Repeat step 1) to determine the change of the GPRC5D-related disease state in the subject, and determine the therapeutic effect of the drug based on the change.
The method according to claim 45, wherein the GPRC5D-associated disorder is cancer or an autoimmune disease, and the cancer is preferably a plasma cell malignancy, such as multiple myeloma, or a B cell malignancy, such as Hodgkin's lymphoma or non-Hodgkin's lymphoma.
The method of claim 46, wherein the cancer is a plasma cell malignancy or a B cell malignancy.
The method of claim 47, wherein said plasma cell malignancy disease is multiple myeloma and said B cell malignancy disease is Hodgkin's lymphoma or non-Hodgkin's lymphoma.
The pharmaceutical composition according to claim 30, used for treating GPRC5D related diseases.